Method for proliferation of antigen-specific T cells

ABSTRACT

The present invention relates to an in vitro method for priming genetically modified T cells suitable for administration to a patient having a tumor. The invention is also directed to the composition obtained by the method and uses thereof.

This application is a continuation of U.S. application Ser. No.14/110,901, filed on 18 Dec. 2013, with is the U.S. national phase ofInternational Application No. PCT/EP2012/056661 filed on 12 Apr. 2012,which claims the benefit of U.S. Provisional Application No. 61/474,904,filed on 13 Apr. 2011, the entire contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to the field of immunology and cancertherapy and more specifically to a method of activation of geneticallymodified antigen specific T cells and the genetically modified T cellsproduced by said method.

BACKGROUND

T cells recognize tumors or infected cells and prevent onset of diseaseby killing these target cells. However, the interplay of tumors orpathogens and the immune system is complex, as demonstrated by cancer orchronic infections developing in the presence of specific T cells,whereby the pathogens or tumors obviously could evade T-cellsurveillance.

The ability of T cells to detect virtually any pathogenic invader isgranted by its extraordinarily diverse receptor repertoire, which allowsthe T-cell pool to recognize a vast number of peptides upon presentationby major histocompatibility complex (MHC) molecules. Still, signalingthrough the T-cell receptor (TCR) (signal 1) is not sufficient foradequate T-cell activation, as costimulatory molecules provideindispensable signals for proliferation, survival, and differentiation(signal 2). In fact, naive T cells that only receive signal 1 withoutsignal 2 are rendered anergic (unresponsive) or die through apoptosis.The integration of signals 1 and 2 is required for full T-cellactivation, and the strength of these signals shapes the size of theensuing T-cell pool. Moreover, full differentiation into effector Tcells is generally dependent on a third signal, which is supplied by theantigen-presenting cell (APC) in soluble form and provides instructivesignals for the type of effector T cell that is required. This‘three-signal’ concept depicts a model for the activation of naive Tcells and the subsequent formation of effector T cells. Yet, the immunesystem provides a plethora of diverse costimulatory molecules and thesevarious types of signal 2 and 3 all contribute in their own uniquemanner to the quality of the T-cell response. Costimulatory signals andsoluble forms of signal 3 can act on particular aspects of T-cellactivation, such as survival, cell cycle progression, type of effectorcell to be developed, and differentiation to either effector or memorycell.

It is now generally accepted that mature antigen-presenting dendriticcells (DCs) have to be “helped” by other lymphocytes, including CD4+ Tcells NK cells and NKT cells, in order to induce long-lived memory CD8+T cells. This “help” induces the mature DCs to differentiate further, aprocess known as licensing. “Helper” signals has multiple effects onDCs, including the upregulation of costimulatory molecules, thesecretion of cytokines, and the upregulation of several antiapoptoticmolecules, all of which cumulatively potentiate the ability of DCs tooptimally activate cognate T cells, especially CD8⁺ T cells. Moreover,“helper” lymphocytes may also express or secrete factors that directlyaffect T cell survival, cell cycle progression, type of effector cell tobe developed, and differentiation to either effector or memory cell.

One strategy for fighting chronic infections or aggressive cancer isadoptive T-cell therapy, which involves the transfer of effector T cellsto restore specific T-cell responses in the host. Recent technicaldevelopments to obtain T cells of wanted specificities have createdincreasing interest in using adoptive T-cell therapy in differentclinical settings. Adoptive cell transfer therapy is the administrationof ex vivo activated and expanded autologous tumor-reactive T cells.There are several potential advantages with the use of adoptive celltransfer therapy in cancer treatment. Tumor specific T cells can beactivated and expanded to large numbers ex vivo, independently of theimmunogenic properties of the tumor, and functional and phenotypicqualities of T cells can be selected prior to their adoptive transfer.

After adoptive transfer, several events must occur for T cells to causethe regression of established tumors. More specifically: —T cells mustbe activated in vivo through antigen specific restimulation, —the Tcells must then expand to levels capable of causing the destruction ofsignificant tumor burdens, —antitumor cells must survive long enough tocomplete the eradication of all tumor cells.

Previously, the criterion used to selecting cells for adoptive transferto patients with solid tumors was the ability of the antitumor T cellsto release IFN-γ and kill tumor cells upon coculture. However, it is nowclear that these criteria alone do not predict in vivo efficacy.Gattinoni et al., J. Clin. Invest. 115:1616-1626 (2005), found that CD8⁺ T cells that acquire complete effector properties and exhibitincreased antitumor reactivity in vitro are less effective at triggeringtumor regressions and cures in vivo. Methods according to prior artrequires restimulation one or more times to reach clinically relevantlevels of tumor specific cytotoxic T cells. See for example Ho et al.(Journal of Immunological Methods, 310(2006), 40-50) and Gritzapis etal. (J. Immunol., 2008; 181; 146-154) wherein restimulation 1-2 timeswere necessary to reach a level of tumor specific CD8+ T cells of about19%. Restimulation makes the cells less active and closer to apoptosis.

The transfer of genes into primary human lymphocytes permits theintroduction of tumor antigen receptor molecules that can endow theengineered cell with antitumor specificity (Vera et al., Curr Gene Ther.2009; 9:396-408; Sadelain et al., Nat Rev Cancer. 2003; 3:35-45; Murphyet al., Immunity. 2005; 22:403-414.). Autologous peripheral bloodlymphocytes (PBLs) can be modified to express a tumor antigen-reactiveT-cell receptor (TCR). Yang et al., (J. Immunother., 2010, vol. 33;648-658) discloses a method of generating antitumour T cells by in vitrotransduction. They use a lentiviral mediated system to geneticallymodify CD8⁺ T cells to express antitumor T-cell antigen receptors(TCRs). In order to efficiently expand CD8⁺ T cells, a rapid expansion(REP) protocol (Ridell et al, U.S. Pat. No. 5,827,642; 1998), consistingof irradiated feeder cells from allogeneic peripheral blood mononuclearcells (PBMC) plus anti-CD3 antibody, was used. However, even if highlyefficient in expansion of T cells in vitro, the REP protocol usuallyinduces T cells with sub-optimal ability to survive and expand afterreinfusion (Robbins et al, Journal of Immunology, 2004, 173:7125-30).

There is a therefore a great need for a method of preparing a T cellpopulation for use in adoptive immunotherapy that increasesproliferation and survival of antigen-specific T cells after reinfusion.

SUMMARY

The present invention relates to an in vitro method for priming ofgenetically modified antigen specific CD4+ and/or CD8+ T cells suitablefor administration to a patient having a tumor. The method comprisesco-culturing antigen receptor expressing target T cells from the patientto be treated, dendritic cells, anti-CD3 antibodies and lymphocytes thathave been sensitized against MHC class I and/or MHC class II antigens onantigen presenting cells (APCs).

The present invention also relates to the antigen specific CD4+ and/orCD8+ T cells obtainable by the method and uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that lymphocytes that have been sensitized againstMHC antigens expressed on irradiated allogeneic peripheral bloodmononuclear cells (PBMCs) in a conventional MLR (=allo-sensitizedallogeneic lymphocytes; ASALs) markedly enhance the expression of CD70on co-cultured mature monocyte-derived DCs which are autologous withrespect to the irradiated PBMCs that were used for priming of ASALs.

FIG. 2 illustrates that gamma-irradiated ASALs enhance the expression ofCD70 on co-cultured mature monocyte-derived DCs which are autologouswith respect to the irradiated PBMCs used for priming of ASALs.

FIG. 3 illustrates that co-culture of ASALs with monocyte-derived DCswhich are autologous with respect to the irradiated PBMCs used forpriming of ASALs induce substantial IL-12 production.

FIG. 4 illustrates that co-culture of ASALs with monocyte-derived DCswhich are autologous with respect to the irradiated PBMCs used forpriming of ASALs induce substantial IFN-gamma production.

FIG. 5 illustrates that co-culture of ASALs with monocyte-derived DCswhich are autologous with respect to the irradiated PBMCs used forpriming of ASALs, induce substantial IL-2 production.

FIG. 6 illustrates the production of IL-2, IL-12 and IFN-gamma and as aresult of co-culture of mature monocyte-derived DCs with ASALs that havebeen depleted of CD4⁺, CD8⁺ or CD56⁺ lymphocytes.

FIG. 7 illustrates that co-culture of ASALs with monocyte-derived DCs,which are autologous with respect to the irradiated PBMCs that were usedfor priming of ASALs, increase the proliferative response innon-sensitized allogeneic CD8⁺ T cells.

FIG. 8 illustrates that addition of irradiated ASALs to monocyte-derivedDCs, which are autologous with respect to the irradiated PBMCs used forpriming of ASALs, during primary stimulation of allogeneic CD8⁺ targetcells leads to increased numbers of CD27-expressing alloreactive CD8⁺target cells when these target cells are restimulated with B-cells thatare autologous with respect to the DCs used for primary target cellstimulation.

FIG. 9 illustrates that addition of irradiated ASALs to irradiatedPBMCs, which are autologous with respect to the irradiated PBMCs usedfor priming of ASALs during primary stimulation of allogeneic CD8⁺target cells leads to decreased numbers of apoptotic(Annexin-V-positive) target cells when these target cells arerestimulated with B-cells that are autologous with respect to the DCsused for primary target cell stimulation.

FIG. 10 illustrates that addition of irradiated ASALs to irradiatedmonocyte-derived DCs, which are autologous with to the irradiated PBMCsused for priming of ASALs, during primary stimulation of allogeneic CD8⁺target cells leads to a stronger (6-fold) secondary proliferativeresponse when these alloreactive CD8⁺ target cells are restimulated withB-cells that are autologous with respect to the DCs used for primarytarget cell stimulation.

FIG. 11 illustrates that addition of irradiated ASALs to irradiatedmonocyte-derived DCs, which are autologous with respect to theirradiated PBMCs used for priming of ASALs, during primary stimulationof allogeneic CD8⁺ target cells leads to a substantial increase ofIFN-gamma production when these alloreactive CD8⁺ target cells arerestimulated with B-cells that are autologous with respect to the DCsused for primary target cell stimulation.

FIG. 12 illustrates that addition of irradiated ASALs to mature,monocyte-derived, DCs which are autologous with respect to theirradiated PBMCs used for priming of ASALs, enhance the number of DCsexpressing CD64 which is the Fc-gamma receptor for anti-CD3 antibodies.

FIG. 13 illustrates that the ASAL protocol, in which CD3+ target T cells(non-transfected) are co-cultured and expanded with mature DCs andirradiated ASALs in medium supplemented with OKT3 (anti-CD3 antibody)and IL-2 for 12 days, induce T cells that are more resistant toapoptosis-inducing H₂O₂ treatment as compared to T cells expanded withthe REP protocol

FIG. 14 illustrates that the ASAL protocol, in which CD3+ target T cells(non-transfected) are co-cultured and expanded with mature DCs andirradiated ASALs in medium supplemented with OKT3 (anti-CD3 antibody)and IL-2 for 12 days, induce T cells that are more resistant toapoptosis-inducing doxorubicin treatment as compared to T cells expandedwith the REP protocol.

FIG. 15 illustrates efficient lentiviral transfection of CD3+ T cells(>40% transfected cells) with a chimeric antigen receptor (CAR) againstthe GD2 antigen expressed on glioblastoma cancer cells

FIG. 16 illustrates that the ASAL protocol induce a similar expansion ofCAR-transfected CD3+ T cells as compared to the standard REP protocol.

FIG. 17 illustrates that the ASAL protocol preferentially expandsCAR-transfected CD8+ T cells as compared to the REP protocol.

FIG. 18 illustrates that the ASAL protocol induce a similar or highernumber of CD27-expressing CAR-transfected CD3+ T cells as compared tothe REP protocol.

FIG. 19 illustrates that the ASAL protocol expands CAR-transfected Tcells with a specific killing ability of GD2-expressing tumor cells thatis similar to the specific killing ability of CAR-transfected T cellsexpanded with the REP protocol. Irrelevant T2 cells were used as controltargets. Diamond (♦)=REP vs. GD2-negative targets; quadrant (▪)=ASAL vs.GD2-negative targets; triangle (▴)=REP vs. GD2-expressing target cells;Cross (X)=ASAL vs. GD2-expressing targets.

FIG. 20 illustrates that the tumor-specific cytotoxic activity inCAR-transfected T cells, as measured by membrane expression of CD107a(marker of lytic granule exocytosis), is more resistant to potentialtumor-derived suppressive factors (IL-10, TNF-beta and/or H₂O₂) when theCAR-transfected T cells have been expanded with the ASAL protocol ascompared with CAR-transfected T cells expanded with REP protocol. Blackcolumn=T cells expanded with REP; White column=CAR transfected T cellsexpanded with the ASAL protocol.

FIG. 21 illustrates that proliferative response of CAR-transfected Tcells, after interaction and killing of target cells, is higher and moreresistant to exposure to potential tumor-derived suppressive factorswhen compared to CAR-transfected T cells that have been expanded withREP protocol. A. No immunosuppressant; B: IL-10/TGF-beta treatment; C.H₂O₂ treatment; D: IL-10/TGF-beta and H₂O₂ treatment

DEFINITIONS

Before the present invention is described, it is to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Also, the term “about” is used to indicate a deviation of +/−2% of thegiven value, preferably +/−5%, and most preferably +/−10% of the numericvalues, where applicable.

In the context of the present invention the term “antigen-specific”relates to the specific recognition/binding by a unique T cell receptor(TCR) of a short unique peptide sequence presented on a self MHCmolecule.

In the context of the present invention the term “priming” and“activation” relates to a programmed activation process that occurs in anaïve antigen-specific T cell that become stimulated byantigen-presenting cells with or without concurrent presence of “helper”cells.

In the context of the present invention the term “responder cells”relates to different lymphocyte subpopulations, including, but notlimited to, T cells, NK cells and NKT cells which respond to co-culturedallogeneic PMBCs by activation and/or proliferation.

In the context of the present invention the term “sensitized cells”relates to different lymphocyte subpopulations, including T cells, NKcells and NKT cells which have been pre-activated by co-culturedallogeneic cells, including PBMCs.

In the context of the present invention the term “target cells” relatesto CD4⁺ or CD8⁺ T cells that become stimulated by either allogeneic orautologous APCs in combination with antibodies, such as anti-CD3antibodies. Sites of patient lymphocyte (target cell) collection can,for example, be peripheral blood, tumor, tumor-draining lymph node orbone marrow.

In the context of the present invention the term “mature” in relation tomonocyte-derived DCs relates to their expression of “maturity-markers”,including, but not limited to, CD40, CD86, CD83 and CCR7 that is inducedby the stimulation of immature DCs with microbial products such as LPSor inflammatory mediators such as TNF-alpha and/or IL-1 beta.

Immature DCs are cells characterized by high endocytic activity and lowT-cell activation potential. Immature DCs constantly sample thesurrounding environment for pathogens such as viruses and bacteria.Immature DCs phagocytose pathogens and degrade their proteins into smallpieces and upon maturation present those fragments at their cell surfaceusing MHC molecules. Simultaneously, they upregulate cell-surfacereceptors that act as co-receptors in T-cell activation such as CD80,CD86, and CD40 greatly enhancing their ability to activate T-cells. Theyalso upregulate CCR7, a chemotactic receptor that induces the dendriticcell to travel through the blood stream to the spleen or through thelymphatic system to a lymph node. Here they act as antigen-presentingcells: they activate helper T-cells and killer T-cells as well asB-cells by presenting them with antigens derived from the pathogen,alongside non-antigen specific costimulatory signals. Mature DCsprobably arise from monocytes, white blood cells which circulate in thebody and, depending on the right signal, can turn into either DCs ormacrophages. The monocytes in turn are formed from stem cells in thebone marrow. Monocyte-derived DCs can be generated in vitro fromperipheral blood monocytes.

In the context of the present invention the term “non-proliferative” ofa cell is used to indicate that the cell has been rendered incapable ofcell division to form progeny. The cell may nonetheless be capable ofresponse to stimulus, or biosynthesis and/or secretion of cell productssuch as cytokines. Methods of making cells non-proliferative are knownin the art. Preferred methods of making cells non-proliferative aretreatment with anti-proliferative drugs such as mitomycin C, orirradiation, such as gamma irradiation. Cells that have been fixed orpermeabilized and are incapable of division are also examples ofnon-proliferative cells.

In the context of the present invention the term “mixed lymphocytereaction”, mixed lymphocyte culture”, “MLR”, and MLC are usedinterchangeably to refer to a mixture comprising a minimum of twodifferent cell populations that are allotypically different. At leastone of the allotypically different cells is a lymphocyte. The cells arecultured together for a time and under suitable conditions to result inthe stimulation of the lymphocytes. A frequent objective of an MLR is toprovide allogeneic stimulation such as may initiate proliferation of thelymphocytes; but unless indicated, proliferation during the culture isnot required. In the proper context, these terms may alternatively referto a mixture of cells derived from such a culture.

As used herein, the term “treatment” refers to clinical intervention inan attempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects include preventingoccurrence or recurrence of disease, alleviation of symptoms, anddiminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, lowering the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

The terms “antigen-presenting cell (s)”, “APC” or “APCs” include bothintact, whole cells as well as other molecules (all of allogeneicorigin) which are capable of inducing the presentation of one or moreantigens, preferably in association with class I MHC molecules, and alltypes of mononuclear cells which are capable of inducing an allogeneicimmune response. Preferably whole viable cells are used as APCs.Examples of suitable APCs are, but not limited to, whole cells such asmonocytes, macrophages, DCs, monocyte-derived DCs, macrophage-derivedDCs, B cells and myeloid leukemia cells e. g. cell lines THP-1, U937,HL-60 or CEM-CM3. Myeloid leukaemia cells are said to provide so calledpre-monocytes.

The terms “cancer”, “neoplasm” and “tumor” are used interchangeably andin either the singular or plural form, as appearing in the presentspecification and claims, refer to cells that have undergone a malignanttransformation that makes them pathological to the host organism.Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e. g. by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Non-limitingexamples of tumors/cancers relevant for the present invention arecarcinomas (e.g. breast cancer, prostate cancer, lung cancer, colorectalcancer, renal cancer, gastric cancer and pancreatic cancer), sarcomas(e.g. bone cancer and synovial cancer), neuro-endocrine tumors (e.g.glioblastoma, medulloblastoma and neuroblastoma), leukemias, lymphomasand squamous cell cancer (e.g. cervical cancer, vaginal cancer and oralcancer). Further, non-limiting examples of tumors/cancers relevant forthe present invention are, glioma, fibroblastoma, neurosarcoma, uterinecancer, melanoma, testicular tumors, astrocytoma, ectopichormone-producing tumor, ovarian cancer, bladder cancer, Wilm's tumor,vasoactive intestinal peptide secreting tumors, head and neck squamouscell cancer, esophageal cancer, or metastatic cancer. Prostate cancerand breast cancer are particularly preferred.

In the context of the present invention the term “culturing” refers tothe in vitro propagation of cells or organisms in media of variouskinds. It is understood that the descendants of a cell grown in culturemay not be completely identical (morphologically, genetically, orphenotypically) to the parent cell. A suitable culturing medium can beselected by the person skilled in the art and examples of such media areRPMI medium or Eagles Minimal Essential Medium (EMEM).

The terms “major histocompatibility complex” and “MHC” refer to acomplex of genes encoding cell-surface molecules that are required forantigen presentation to T cells and for rapid graft rejection. Inhumans, the MHC complex is also known as the HLA complex. The proteinsencoded by the MHC complex are known as “MHC molecules” and areclassified into class I and class II MHC molecules. Class I MHCmolecules include membrane heterodimeric proteins made up of a chainencoded in the MHC associated non-covalently with β2-microglobulin.Class I MHC molecules are expressed by nearly all nucleated cells andhave been shown to function in antigen presentation to CD8+ T cells.Class I molecules include HLA-A, -B, and -C in humans. Class I moleculesgenerally bind peptides 8-10 amino acids in length. Class II MHCmolecules also include membrane heterodimeric proteins.

Class II MHCs are known to participate in antigen presentation to CD4+ Tcells and, in humans, include HLA-DP, -DQ, and DR. Class II moleculesgenerally bind peptides 12-20 amino acid residues in length. The term“MHC restriction” refers to a characteristic of T cells that permitsthem to recognize antigen only after it is processed and the resultingantigenic peptides are displayed in association with either a self classI or self class II MHC molecule.

The terms “vaccine”, “immunogen”, or immunogenic composition” are usedherein to refer to a compound or composition that is capable ofconferring a degree of specific immunity when administered to a human oranimal subject. As used in this disclosure, a “cellular vaccine” or“cellular immunogen” refers to a composition comprising at least onecell population, which is optionally inactivated, as an activeingredient. The immunogens, and immunogenic compositions of thisinvention are active, which mean that they are capable of stimulating aspecific immunological response (such as an anti-tumor antigen oranti-cancer cell response) mediated at least in part by the immunesystem of the host. The immunological response may comprise antibodies,immunoreactive cells (such as helper/inducer or cytotoxic cells), or anycombination thereof, and is preferably directed towards an antigen thatis present on a tumor towards which the treatment is directed. Theresponse may be elicited or restimulated in a subject by administrationof either single or multiple doses.

A compound or composition is “immunogenic” if it is capable of either:a) generating an immune response against an antigen (such as a tumorantigen) in a naive individual; or b) reconstituting, boosting, ormaintaining an immune response in an individual beyond what would occurif the compound or composition was not administered. A composition isimmunogenic if it is capable of attaining either of these criteria whenadministered in single or multiple doses.

Description

The present invention relates to the production of allo-sensitizedallogeneic lymphocytes (ASALs) to promote increased proliferation andsurvival of antigen-specific T cells during their activation byantigen-presenting cells, including dendritic cells (DCs) in combinationwith anti-CD3 antibodies.

The present invention is based on in vitro studies using peripheralblood mononuclear cells (PBMCs), and subpopulations thereof, from humanhealthy blood donors where a positive regulatory role for ASALs in theinduction of antigen-specific human CD8⁺ T cell responses wasdemonstrated. Using an allogeneic in vitro model, tracking proliferationand survival of alloreactive CD8⁺ T cells in the presence of ASALs, theproliferative capacity after re-stimulation was increased more that5-fold and apoptotic cell death reduced from 10 to 5%.

Antigen-specific human CD4⁺ and CD8⁺ T cells can be generated in vitrothrough transduction or transfection of genes encoding tumor antigenreceptors. The present invention relates to a method of preparing a Tcell population for use in adoptive immunotherapy comprising T cellsengineered (by viral transduction, transfection, electroporation orother methods of introducing genetic material) to express a T cellreceptor (TCR) or a chimeric antigen-receptor (CAR) that recognize thetarget antigen; activating these engineered T cells with DCs in thepresence of sensitized allogeneic lymphocytes and anti-CD3 antibodies;expanding these cells in culture; and reintroducing these cells backinto the patient.

Addition of ASALs leads to a strongly up regulated expression of theco-stimulatory molecule CD70 on antigen-presenting DCs and to productionof IL-12 and IFN-gamma, two factors with a well-known positive impact onT cell commitment into type 1 CD4+ and CD8+ T cells. Further, additionof ASALs also led to production of IL-2, a well-known growth factor forT cells. Notably, CD70-mediated interactions have recently been shown topromote survival of activated T cells throughout successive rounds ofdivision and thereby contribute to the accumulation of effector T cells.

More specifically, the present invention relates to an in vitro methodfor priming of genetically modified antigen specific CD4+ and/or CD8+ Tcells suitable for administration to a patient having a tumor. Themethod comprises co-culturing tumor antigen receptor expressing target Tcells from the patient to be treated with DCs, anti-CD3 antibodies andlymphocytes sensitized against MHC class I and/or MHC class II antigenson antigen presenting cells (APCs), wherein the APCs preferably beingallogeneic with respect to the lymphocytes and the dendritic cellspreferably monocyte derived. The addition of anti-CD3 antibodies willlead to their binding to the dendritic cells by Fc/Fc-receptorinteractions that enable the antibody-armed dendritic cells to directlyinteract and activate CD3-expressing T cells.

Target T cells can be transformed with T cell receptor (TCR) codinggenes or alternatively through the use of a chimeric antigen receptor(CAR) that is capable of relaying excitatory signals to T cells in anon-MHC-restricted manner. These hybrid proteins, composed of anextracellular antigen recognition domain fused to an intracellularT-cell activation domain, may therefore be used in patients regardlessof their human leukocyte antigen genotype. Prior to genetictransformation, the target T cells are preferably pre-stimulated withanti-CD3 antibodies in order to optimize subsequent transformation. Anysuitable CAR can be used in the present invention. The CAR ligand shouldbe expressed by the tumor cell. Selecting and preparing a suitable CARis within the skills of the person skilled in the art. Non-limitingexamples CAR ligands are VEGFR-2 (vascular endothelial growth factorreceptor-2), Her2/neu, CEA (carcino embryonic antigen), CD19, CD20 andGD2 (ganglioside antigen).

Genetic transformation of TCR- or CAR-coding genes into T cells can beperformed by using any suitable method known to the skilled person, suchas transfection or transduction (see for example Sambrook et al.,Molecular Cloning, A Laboratory Manual, 3rd ed., vol. 1-3, Cold. SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). Transfection can be madeusing viral vectors. Non-limiting examples of viral vectors includeretro, lenti, adeno, adeno-associated viral vectors. Transductionincludes but is not limited to electrotransfer of plasmids,transposon/transposase systems and micro-injection

The ASALs are responder cells obtained from a mixed leukocyte reactionand are subsequently cultured together with dendritic cells and targetcells in a culture medium containing anti-CD3 antibodies. The ASALs canbe autologous or allogeneic with respect to the patient and with respectto the dendritic cells. The dendritic cells can be autologous orallogeneic with respect to the patient to be treated and with respect tothe stimulator cells used for primary MLR-induced activation of ASALs.The ASALs are selected from the group consisting of peripheral bloodlymphocytes, including CD4⁺ T cells, CD8⁺ T cells and natural killer(NK) cells. The target cells are CD4⁺ and/or CD8⁺ T cells.

In the inventive method the CD3 antibodies will act as unspecificstimulator of the transfected T cells (the T cells will become activatedwhen the anti-CD3 antibody binds to the CD3 molecule on the T cell. Thetransfected T cells and the dendritic cells are autologous orallogeneic. The CD3 antibodies will also restimulate the ASALs meaningthat the ASALs can be autologous or allogeneic with respect to thedendritic cells. Preferably, the dendritic cells and the ASALs areallogeneic. The dendritic cells and the ASALs are also preferably, butnot necessarily, allogeneic (i.e. from healthy blood donors) withrespect to the transfected patient T cells

Addition of ASALs further leads to an enrichment of a population oftarget CD8⁺ T cells expressing high levels of CD27. CD27⁺ CD8⁺ T cellsrepresent potentially more effective CTLs (cytotoxic T cells) foradoptive immunotherapy since they can provide an antigen-drivenautocrine signal for proliferation. Such helper-independent CD8+ T cellswould not require exogenous help in the form of IL-2 or CD4⁺ T cells tosurvive and expand. Thus, the present invention provides methods fortreating an immune-mediated disease by providing a subject with a CD8⁺ Tcell population that is programmed for strong cytotoxic activity in theabsence or reduced presence of additional cytokines, such as IL-2, orCD4⁺ T cells. The methods are particularly useful for ex vivo expansionof cytolytic, antigen-specific CD8+ T cells, but may also be used forexpansion of tumor-specific CD4⁺ T cells.

The percentage of cytolytic antigen-specific CD8+ T cells expressed aspercentage of the total number of CD8+ T lymphocytes is preferably atleast about 5%, more preferably at least about 10%, more preferably atleast about 15%, more preferably at least about 20%, even morepreferably at least about 25%, even more preferably at least about 30%and most preferably at least about 35%.

Both CD8+ and CD4+ T cells are needed for efficient cytotoxicity. TheCD8+ T cells are cytotoxic while the CD4+ T cells release growthproliferating factors, such as IL-2. Preferably the number CD8+ T cellsexceeds the number of CD4+ T cells since expansion protocols that favorexpansion of CD4+ T cells over CD8+ T cells are expected to compromisein vivo anti-tumor efficacy (Yang et al. Journal of Immunotherapy 2010;33:648.

More specifically, the method of the present invention relates to an invitro method for priming of TCR- or CAR-transformed antigen specificCD4+ and/or CD8+ T cells suitable for administration to a patient havinga tumor, said method comprising the following steps:

a) culturing non-proliferating antigen presenting cells from the patientor from a healthy donor together with peripheral blood mononuclear cellsthat are allogeneic with respect to the antigen presenting cells,

b) culturing monocytes, from the patient or from a healthy donor, in acomposition allowing the monocytes to mature to mature DCs. (thecomposition is further described below), and

c) culturing allo-sensitized lymphocytes, including but not limited toCD4⁺ T cells, CD8⁺ T cells and/or natural killer (NK) cells from step a)with mature DCs from step b) together with TCR- or CAR-transformedtarget cells, including CD4+ T cells and CD8+ T cells, in a culturemedium containing anti-CD3 antibodies.

Monocyte-derived DCs are obtained by first culturing monocytes in acomposition comprising GM-CSF and IL-4 for about 2-7 days, preferablyabout 5 days to obtain immature DCs and subsequently add a secondcomposition that enables the immature DCs to become mature DCs byculturing for at least about 12 to 72 hours and preferably about 24-48hours. The second composition comprises components that allow theimmature DCs to become mature monocyte-derived DCs that can be used toactivate CD4+ and CD8+ T cells. In one embodiment the second compositioncomprises TNF alfa, IL-1 beta, interferon gamma, interferon beta and aTLR3 ligand, such as poly-I:C (Mailliard et al., Alpha-type-1 polarizedDCs: a novel immunization tool with optimized CTL-inducing activity.Cancer Res. 2004; 64:5934-5937.). In another embodiment the secondcomposition comprises interferon gamma, a TLR 3 and/or a TLR 4 ligandand a TLR7 and/or a TLR 8 ligand and/or a TLR9 ligand. Non-limitingexamples of a TLR 3 ligand is poly-I:C, of a TLR7/8 ligand is R848, andof a TLR9 ligand is CpG.

The sensitization of allogeneic lymphocytes is induced by a traditionalmixed leukocyte reaction (MLR or MLC—mixed leukocyte culture) comprisingculturing inactivated antigen presenting cells with peripheral bloodmononuclear cells (PBMCs) from a healthy donor, wherein the antigenpresenting cells are allogeneic with respect to the lymphocytes. Theperformance of an MLR is well known to the skilled person (Jordan W J,Ritter M A. Optimal analysis of composite cytokine responses duringalloreactivity. J Immunol Methods 2002; 260: 1-14. In an MLR PBMCs(mainly lymphocytes) from two individuals are mixed together in tissueculture for several days. Lymphocytes from incompatible individuals willstimulate each other to proliferate significantly (measured for exampleby tritiated thymidine uptake) whereas those from compatible individualswill not. In a one-way MLC, the lymphocytes from one of the individualsare pre-treated with anti-proliferative drugs such as mitomycin or withirradiation, thereby allowing only the untreated lymphocytes from theother individual to proliferate in response to foreignhistocompatibility antigens.

The antigen presenting cells used in the MLR are selected from the groupconsisting of PBMCs and monocytes-derived DCs.

In the method of the present invention the cells (tumor antigen receptorexpressing target T cells from the patient to be treated, dendriticcells, anti-CD3 antibodies and lymphocytes that have been sensitizedagainst MHC class I and/or MHC class II antigens on antigen presentingcells (APCs)) are co-cultured for about 20 days, preferably for about 4to 20 days, preferably 6 to 20 days, more preferably 7 to 14 days andmost preferably about 9 to 14 days.

Exogenous IL-2, IL-7, IL-15, anti-IL-4 and/or IL-21 can be added to thecell culture in order to optimize cell proliferation and survival.

It is also possible to restimulate the primed antigen specific CD4+and/or CD8+ T cells by culturing said cells together with new DCs,anti-CD3 antibodies and new sensitized lymphocytes that have beenactivated by non-proliferating antigen presenting cells that areallogeneic with respect to the lymphocytes, and optionally addition ofexogenous IL-2, IL-7, IL-15, anti-IL-4 and/or IL-21 to the cell culture.

The present invention also relates to an immunogenic compositionobtainable by the method described above as well as the antigen specificCD4+ and/or CD8+ T cells obtainable by the method described above.

The antigen specific CD4+ and/or CD8+ T cells are suitable foradministration to a patient and preferably have at least one of thefollowing features:

-   -   ability to proliferate    -   express low levels of the apoptosis marker Annexin-V (i.e. no        more than 40%, preferably no more than 20%, of the cells should        exhibit positive staining for Annexin-V by FACS determination)    -   express CD27 and/or CD28 at their cell surface

A further ability of the specific CD4+ and/or CD8+ T cells obtainable bythe method of the invention is the ability to be activated by, and/orkill tumor cells or tumor-loaded antigen-presenting cells that areMHC-compatible with respect to the antigen specific T cells in vitro.The specific CD4+ and/or CD8+ T cells obtainable by the method of theinvention also possess the ability to kill relevant tumor target cellsin vitro despite preculture with immunosuppressive factors such asIL-10, TGF-beta and/or H₂O₂, have the ability to proliferate afterkilling of relevant tumor target cells in vitro as well as ability toproliferate after killing of relevant tumor target cells in vitrodespite preculture with immunosuppressive factors such as IL-10,TGF-beta and/or H₂O₂.

The present invention relates to the antigen specific CD4+ and/or CD8+ Tcells obtained by the inventive method for use as a medicament and foruse of said the antigen specific CD4+ and/or CD8+ T cells for themanufacture of a medicament.

Further, the present invention relates to the use of antigen specificCD4+ and/or CD8+ T cells obtainable by the method of the invention or asdefined above for use in the treatment of a tumor or for eliciting ananti-tumor immunological response in a human as well as for themanufacture of a medicament for the treatment of a tumor or foreliciting an anti-tumor immunological response in a human. The CD4+and/or CD8+ T cells can be administered after the first stimulation oralternatively after restimulation. In one embodiment the CD4+ and/orCD8+ T cells are administered in combination with a therapeutic cancervaccine.

Methods of using T cell populations for adoptive cell therapy intreatment of human subjects are known to clinicians skilled in the art.T cell populations prepared according to the methods described hereinand known in the art can be used in such methods. For example, adoptivecell therapy using tumor-infiltrating lymphocytes, with MART-I antigenspecific T cells have been tested in the clinic (Powell et al., Blood105:241-250, 2005). Patients with renal cell carcinoma have beenvaccinated with irradiated autologous tumor cells. Harvested cells weresecondarily activated with anti-CD3 monoclonal antibody and IL-2 andthen re-administered to the patients (Chang et al., J. Clinical Oncology21:884-890, 2003.)

Antigen-primed T cells undergo increased proliferation and decreasedapoptosis upon re-stimulation when exposed to ASALs during their initialDC-mediated priming in vitro. Thus, methods for enhancing secondary Tcell responses upon vaccination if adoptively transferred back to thepatient before and/or during vaccination are also contemplated by thepresent invention.

The present invention also provides methods for improving cancer vaccinetherapy. Many tumors express foreign antigens that can potentially serveas targets for destruction by the immune system. Cancer vaccinesgenerate a systemic tumor-specific immune response in a subject thatcomprises both humoral and cellular components. The response is elicitedfrom the subject's own immune system by administering a vaccinecomposition at a site distant from the tumor or at the site of alocalized tumor. The antibodies or immune cells bind the tumor antigenand lyse the tumor cells. However, there remains a need for increased Tcell-responsiveness upon vaccination of cancer patients. Adoptivetransfer of preactivated apoptosis-resistant tumor-specific T cells withhigh proliferative potential before, or at the time of vaccination, maytherefore enhance vaccine-mediated immune responses in vivo.

The composition according to the invention can also be administered incombination with a therapeutic cancer vaccine. Non-limiting examples ofsuch therapeutic cancer vaccines are ex vivo-propagated and tumor-loadedDCs, cytokine producing tumor cells, DNA-vaccination and vaccines usingTLR-ligands in combination with tumor antigens.

The cells obtainable by the method of the invention can be administereddirectly to an organism, such as a human, to increase proliferation andsurvival of antigen-specific T cells during their activation.Administration of these cells, often with pharmaceutically acceptablecarriers, is by any of the routes normally used for introducing a cellinto ultimate contact with a mammal's blood or tissue cells.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous and intratumoral routes and carriers include aqueousisotonic sterile injection solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. Intravenousadministration is the preferred method of administration for the CD4+and/or CD8+ T cells of the invention.

The dose of the CD4+ and/or CD8+ T cells administered to a patient, inthe context of the present invention should be sufficient to enhance theimmune response in the patient. Thus, cells are administered to apatient in an amount sufficient to elicit an effective immune responseto the tumor antigen and/or to alleviate, reduce, cure or at leastpartially arrest symptoms and/or complications from the disease. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose”. The dose will be determined by the activity of thecells produced and the condition of the patient, as well as the bodyweight or surface area of the patient to be treated. In determining theeffective amount of the cell to be administered in the treatment orprophylaxis of diseases such as cancer the physician needs to evaluateprogression of the disease and the induction of immune response againstany relevant tumor antigens.

There are several major advantages of the invention compared to methodsof the prior art. The present invention provides a high level of tumorspecific CD8+ T cells without the need of restimulation. Restimulationmakes the cells less active and brings them closer to apoptosis. Thus, amethod that efficiently expands tumor specific T cells without the needfor restimulation is an advantage. In addition, without the need torestimulate the cells, the tumor specific T cells can be brought back tothe patient in a shorter period of time and it is more cost efficient.Further, with the use of the method according to the present inventionthere is no need for depletion of suppressor cells or the addition ofexogenous growth factors which are very costly processes. Adoptivetransfer of autologous tumor specific T cells that are cultured fromtumor infiltrating lympohocytes can cause regressions of advanced tumorsin humans but cannot be reliably cultured from most human tumors,Methods have therefore been developed to engineer a large number ofblood-derived T cells to express genes encoding tumor-antigen-specific Tcell receptors. In the case of a chimeric antigen receptor (CAR), it canbe used in patients regardless of their human leukocyte antigengenotype.

Although the expansion efficacy of the inventive method is similar tothe REP protocol, the ASALs expanded with the present method have a muchhigher killing ability in vivo compared to the REP expanded T cells. Themain reason for this is that the REP produced T cells are less resistantto the immunosuppressive environment created by the tumor compared tothe T cells expanded with the method according to the present invention.Further, the inventive method expands CD8+ T cells at a higher ratiocompared to the REP protocol which is beneficial as protocols that favorCD4+ T cell expansion show reduced in vivo anti-tumor efficacy.

Encompassed by the present invention is any combination of the differentaspects and features disclosed.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Material & Methods

Allosensitized allogeneic lymphocytes (ASALs) were generated in astandard one-way mixed leukocyte reaction (MLR) by co-culturing gammairradiated PBMC from a healthy blood donor with non-irradiated PBMCsfrom an allogeneic donor (with respect to the healthy blood donor) at aratio of 1:1 in serum-free X-VIVO 15 medium in tissue culture flasks for5-7 days. For propagation of immature DCs, peripheral blood mononuclearcells (PBMCs) obtained from healthy blood donors were isolated ondensity gradients (Lymphoprep, Nycomed, Oslo, Norway). Isolated PBMCswere resuspended in AIM-V medium (Invitrogen, Paisley, UK), plated in24-well plastic culture plates at 2.5×10⁶ cells per well and allowed toadhere for 2 hours. Non-adherent cells were removed and the remainingadherent monocytes, were cultured in AIM-V medium supplemented withrecombinant human GM-CSF and IL-4 (R&D Systems, Abingdon, UK; both at1,000 U/mL) for 4-6 days. Maturation of immature DC was induced bysupplementing the culture media with IFN-α (3,000 U/mL), IFN-γ (1,000U/mL), TNF-α (50 ng/mL), IL-1β (25 ng/mL) (all from R&D Systems) andp-I:C (Sigma-Aldrich; 20 μg/mL) during the last 24 hours of incubation.

The mature DC populations all contained more than 70% CD83+ DCs asdetermined by FACS analysis.

After washing, mature DCs were co-cultured with non-irradiated orgamma-irradiated (25 Grey) ASALs in X-VIVO 15 medium for 24 h andanalyzed by FACS. Sensitization of alloreactive lymphocytes wasperformed by conducting a primary one-way MLR in serum-free culturemedia (X-VIVO 15) for 5-6 days with gamma-irradiated PBMCs as stimulatorcells and non-irradiated PBMCs as responder cells. PE-conjugatedanti-human CD70 was used for FACS studies.

Results:

As shown in FIG. 1, ASALs markedly enhance the expression of CD70 onmature monocyte-derived DCs which are autologous with respect to theirradiated PBMCs used for priming of ASALs.

As shown in FIG. 2, gamma-irradiated ASALs similarly enhance theexpression of CD70 on mature monocyte-derived DCs which are autologouswith respect to the irradiated PBMCs used for priming of ASALs.

As shown in FIGS. 3, 4 and 5 co-culture of ASALs with mature DCs whichare autologous with respect to the irradiated PBMCs used for priming ofASALs, induce a substantial production of IL-12, IFN-gamma and IL-2.

Example 2 Material and Methods

ASALs were generated during a conventional MLR for 7 days usingirradiated allogeneic PBMCs as stimulators (see Example 1). Afterharvest and irradiation, the bulk population of ASALs (“MLR”) or ASALsdepleted of CD4⁺, CD8⁺ or CD56⁺ (NK/NKT) cells were co-cultured withmature allogeneic monocyte-derived DCs (autologous with respect to thePBMCs used for priming of ASALs). Co-culture supernatants were collectedafter 24 h and subsequently assayed for IL-2, IL-12 and IFN-gammaproduction.

Results:

IL-2 production was found to be strictly CD4-dependent (FIG. 6A), whileIL-12 production (FIG. 6B) showed no ASAL-dependence at all andIFN-gamma production (FIG. 6C) showed partial dependence on co-culturedand alloprimed CD4⁺, CD8⁺ and CD56⁺ (NK/NKT) within the ASAL-population.

Example 3 Material and Methods

Immature DCs were generated by plastic adherence of monocytes. Monocyteswere cultured for 7 days in CellGro® DC supplemented with IL-4 andGM-CFS, both at 1000 U/mL. Maturation of DCs was induced by the additionof 50 ng/mL TNF-α, 25 ng/mL IL-1β, 50 ng/mL IFN-γ, 3000 U/mL IFN-α and20 μg/mL Poly I:C during the last 2 days of incubation.

ASALs were generated in a one-way mixed lymphocyte reaction byco-culturing gamma irradiated allogenic PBMC and non-irradiatedautologous PBMC, with respect to DC donor, at a ratio of 1:1 in X-VIVO15 for 7 days.

CD8⁺ T lymphocytes were isolated by positive selection from autologousPBMC which had been cultured in X-VIVO 15 supplemented with 50 ng/mLIL-15 at a final concentration of 0.5×10⁶ lymphocytes/mL for 7 days.PBMC were centrifuged and re-suspended in PBS-0.5% BSA-2M EDTA at afinal concentration of 1×10⁷/80 μL. PBMC were incubated with CD8⁺MicroBeads (Miltenyi Biotec) for 15 min at 4° C., washed, re-suspendedand placed onto a LS MACS column. Unlabeled cells were washed throughand total effluent containing CD8⁺ lymphocytes were collected. IsolatedCD8⁺ T lymphocytes were resuspended in pre-warmed PBS-1% BSA to aconcentration of 1×10⁶/mL and stained with 10 μM CFSE (Molecular probesInvitrogen) for 10 min at 37° C. Staining was terminated by addition of5 mL ice-cold X-VIVO 15 medium and incubated on ice for 5 min. Cellswere washed twice in medium and re-suspended to a final concentration of1×10⁶/mL. Stained CD8⁺ T lymphocytes were co-cultured for 4-7 days withirradiated allosensitized allogenic PBMC and matured autologous DC at aratio of 4:4:1. Following culture, lymphocytes were harvested andstained with CD3-APC-H7, CD8-PerCP, CD27-APC and Annexin V. Thepercentage of proliferating CD8⁺ T lymphocytes was determined by flowcytometry and expressed as percentage of total lymphocytes.

Results:

Results: As illustrated in FIG. 7, addition of irradiated “AlloHelpers”(=ASALs) strongly increase CD8⁺ T cell divisions (more cells with lowfluorescence intensity=more dots to the left in the dot-plot). ASALsthus augment the ability of monocyted-derived DCs to induce aproliferative response in alloreactive CD8+ T cells.

Example 4 Material and Methods: See M&M in Example 1

CD8+ lymphocytes were isolated (using negative selection withantibody-coated magnetic beads) after co-culture of DCs, irradiatedASALs (allogeneic to the DCs) for 6 days and subsequently restimulatedwith B-cells (autologous to the DCs used during primary stimulation) andstained for expression of CD27 and Annexin-V. Subsequent analysis wasperformed with FACS.

Results:

As shown in FIG. 8, addition of ASALs during primary stimulationsubstantially increased expression of CD27 when the CD8⁺ cells werere-stimulated with B-cells.

Addition of ASALs during primary stimulation substantially reducedexpression of Annexin-V (apoptosis marker) when the CD8+ cells werere-stimulated with B-cells (see FIG. 9) thus making the CD8+ T cellsmore resistant to enter apoptosis.

Example 5 Material and Methods: See M&M in Example 4

Before restimulation with B-cells the primed and isolated CD8⁺ cellswere pulsed with 3H-Thymidine.

Results:

As shown in FIG. 10, addition of ASALs during primary stimulationstrongly increased the proliferative response (as measured byincorporation of 3H-Thymidine, cpm/min, day 3) of alloreactive CD8⁺cells after restimulation.

Example 6 Material and Methods: See M&M in Example 4

After co-culture of B-cells and pre-activated CD8⁺ cells for 2 daysculture supernatant was collected and analyzed for IFN-gamma productionby a conventional ELISA

Results:

FIG. 11 shows that addition of ASALs during primary stimulationsubstantially increased production of IFN-gamma by alloreactive CD8⁺cells after restimulation.

Example 7—Expansion of CAR-Transfected T Cells

Material and Methods:

The basal culture medium consisted of RPMI Media 1640 supplemented with10% human serum, 1% penicillin (100 U/ml), 1% HEPES, 0.5% L-glutamineand 160 μl β mercapto-ethanol.

Immature DCs were generated by plastic adherence of monocytes. Monocyteswere subsequently cultured for 7 days in basal culture mediumsupplemented with IL-4 and GM-CFS, both at 1000 U/mL. Maturation of DCswas induced by the addition of 20 ug/mL Poly-I:C, 2.5 ug/mL R848 and 50ng/mL IFN-gamma during the last 24 hours of incubation.

ASALs were generated in a one-way mixed lymphocyte reaction byco-culturing gamma irradiated PBMC and non-irradiated allogeneic PBMC ata ratio of 1:1 in basal culture medium for 7 days.

T Cell Transfection:

Isolated PBMCs (5×10(5)/m L) were initially activated for 3 days byadding IL-2 (100 IU/mL) and anti-CD3 (OKT3) (50 ng/mL) to the DC media.Thereafter the non-adherent cells (mainly activated T cells) weretransfected with CAR against GD2 (a ganglioside antigen which is highlyexpressed on neuroblastoma cells), by incubation in culture mediumcontaining CAR-lentivirus (20 uL), Sequa-Brene (1 mg/mL) and IL-2 (100IU/mL) during 4 hours. After washing the cells were cultured in mediumsupplied with IL-2 (100 IU/mL) for 5 days. Transfection level wasthereafter analyzed by flow cytometry using PE-A conjugated antibodiesagainst lentiviral-CAR.

T Cell Expansion:

After washing, the CAR-transfected T cells were subsequently expanded inculture medium for 12 days in T-25 flasks using either the ASAL protocolconsisting of mature DCs+irradiated ASALs+CAR-transfected T cells (1:4:1ratio), IL-2 (100 IU/mL) and OKT3 (50 ng/mL) or the standard REPprotocol (Yang et al, Journal of Immunotherapy 2010; 33:648) consistingof irradiated allogeneic PBMCs from 3 different donors+CAR-transfected Tcells (100:1 ratio), IL-2 (100 IU/mL) and OKT3 (50 ng/mL). At day 3, 6and 9, the culture medium was removed and replenished with fresh medium,100 IU/mL IL-2 and 50 ng/mL OKT3 for extended culture.

Flow Cytometry Analysis:

The cells were washed with PBS twice and stained for 15 min at roomtemperature (avoiding light) by using specific fluorophore (fluoresceinisothiocyanate (FITC), APC, or phycoerythrin (PE))-labeled antibodies(BD Biosciences, SanDiego, Calif.) against cell surface markers (CD3,CD4, CD8, CD27 CD64 and CAR (GD2)). The FACS analysis was performed onBD FACS Canto II (BD Biosciences, USA) and the data were analyzed withBD FACS Canto II software.

Apoptosis Assay:

The Annexin V apoptotic assay (BD Biosciences) was used to evaluate theviability of T cells after expansion for 12 days with differentexpansion protocols in control medium and in medium whereapoptosis-inducing agents (H2O2 or doxorubicin) were subsequently addedfor 24 hours. Briefly, T cells were washed with PBS twice andre-suspended in binding buffer and then incubated with AnnexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 minat room temperature, followed by wash and flow cytometry analysis

Measurement of Tumor Cell Killing:

CAR-transfected and expanded (12 days) T cells were co-cultured for 48hours with the GD2-expressing neuroblastoma cell line IMR-3(co-expressing the Luciferase report gene) at different effector:targetcell ratios (E:T ratio) in each well of a round-bottom 96-well plate.The ability of transfected and expanded T cells to lyse relevant tumorcells was evaluated using a luciferase expression assay (Fu et al, PloS1, 2010, 5, e11867). The relative cell viability (%) was calculated asraw light unit (RLU) and normalized to the luciferase activity fromtarget cells (IMR-3) without effector T cells.

Tumor cell killing by T cells exposed to potential tumor-derivedsuppressive factors was also estimated by flow cytometry usingconjugated antibodies against membrane bound CD107a which is a marker oflytic granule exocytosis on effector T cells. T cells were exposed withIL-10 (10 ng/mL) and TGF-beta (2.5 ng/mL) for 4 hours, H202 (25 uM) for24 hours or IL-10 and TGF-beta (both for 4 hours) in combination withH2O2 (24 hours) before co-culture with target cells.

The CellTrace™ CFSE cell proliferation kit (Invitrogen, Eugene, Oreg.,USA) was used to evaluate T-cell proliferation 4 days afterinteraction/killing of target antigen-expressing tumor cells with orwithout prior exposure to potential tumor-derived suppressive factors. Tcells were exposed with IL-10 (10 ng/mL) and TGF-beta (2.5 ng/mL) for 4hours, H202 (25 uM) for 24 hours or IL-10 and TGF-beta (both for 4hours) in combination with H2O2 (24 hours) before co-culture with targetcells.

Results:

As shown in FIG. 12, addition of irradiated ASALs to irradiated, mature,monocyte-derived DCs, which are autologous with respect to theirradiated PBMCs used for priming of ASALs, enhance the number of CD64(Fc-gammaR)-expressing DCs. This receptor is supposed to catch theFc-part of soluble anti-CD3 antibodies. Such anti-CD3-“armed” DCs maythen directly interact with and activate cocultured autologous orallogeneic CD3 expressing T cells.

FIG. 13 shows the viability after H₂O₂ treatment. The ASAL protocolinduces T cells that are more resistant to apoptosis-inducing H₂O₂treatment as compared to T cells expanded with the REP protocol. SinceH₂O₂ is a well-known immunosuppressing factor within tumors it may beexpected that the ASAL protocol will expand T cells with a superiorresistance to tumor-derived H₂O₂ in vivo.

FIG. 14 shows the viability after doxorubicin treatment. The ASALprotocol induces T cells that are more resistant to apoptosis-inducingdoxorubicin treatment as compared to T cells expanded with the REPprotocol. Since doxorubicin is a frequently used anti-cancer drug it maybe expected that the ASAL protocol will expand T cells with a superiorresistance to apoptosis induced by concurrent anti-cancer treatment withdoxorubicin in vivo

CD3+ T cells are efficiently transfected (>40% transfected cells) with achimeric antigen receptor (CAR) against the GD2 antigen (expressed onglioblastoma cancer cells) (see FIG. 15). Left figure=beforetransfection, right figure after transfection. All dots within the upperright quadrant represent CAR-transfected T cells.

The ASAL protocol induces a similar expansion of CAR-transfected CD3+ Tcells as compared to the standard REP protocol (see FIG. 16), butexpands CD8+ T cells at a higher ratio as compared to the REP protocol(see FIG. 17). This finding is of clinical significance since expansionprotocols that favor expansion of CD4+ T cells are expected tocompromise in vivo anti-tumor efficacy (Yang et al. Journal ofImmunotherapy 2010; 33:648)

Further, the ASAL protocol induce a similar or higher number ofCD27-expressing CD3+ T cells as compared to the REP protocol (see FIG.18). This finding is clinically relevant since the persistence of Tcells following adoptive transfer in humans has been shown to bedirectly correlated to high levels of CD27 expression on the reinfused Tcells. CD27⁺ CD8⁺ T cells represent potentially more effective CTLs(cytotoxic T cells) for adoptive immunotherapy since they can provide anantigen-driven autocrine signal for proliferation. Suchhelper-independent CD8+ T cells would not require exogenous help in theform of IL-2 or CD4⁺ T cells to survive and expand.

As shown in FIG. 19, the ASAL protocol expand CAR-transfected T cellswith a specific killing ability that is similar to the specific killingability of CAR-transfected T cells expanded with the REP protocol.

The tumor-specific cytotoxic capacity in CAR-transfected T cells (asmeasured by membrane expression of CD107a) is more resistant to exposureof potential tumor-derived suppressive factors (IL-10, TNF-beta and/orH₂O₂) when the CAR-transfected T cells have been expanded with theASAL-protocol as compared with CAR transfected T cells expanded with REPprotocol (see FIG. 20).

The proliferative response of CAR transfected T cells, after interactionand killing of target cells, is higher and more resistant to potentialtumor-derived suppressive factors when compared to CAR transfected Tcells that have been expanded with REP protocol (a protocol frequentlyused to expand T cells; see Yang et al, Journal of Immunotherapy 2010;33:648). Thus, after killing of target cells the T cells generated bythe inventive method are re-stimulated and may therefore attack and killnew cancer cells

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims that follow. In particular, it is contemplated by theinventor that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims.

We claim:
 1. Antigen specific CD4+ and/or CD8+ T cells obtained by invitro priming of genetically modified antigen specific CD4+ and/or CD8+T cells suitable for administration to a patient having a tumor, whereinthe priming comprises co-culturing tumor antigen receptor expressingtarget T cells from the patient to be treated, mature dendritic cells,anti-CD3 antibodies and lymphocytes that have been sensitized againstWIC class I and/or WIC class II antigens on antigen presenting cells(APCs).
 2. Antigen specific CD4+ and/or CD8+ T cells suitable foradministration to a patient wherein said CD4+ and/or CD8+ T cells: havethe ability to proliferate, no more than 40% of the cells should exhibitpositive staining for Annexin-V, and/or express CD27 and/or CD28 attheir cell surface.
 3. A medicament comprising antigen specific CD4+and/or CD8+ T cells according to claim 1 for use as a medicament.
 4. Amedicament comprising antigen specific CD4+ and/or CD8+ T cellsaccording to claim 2 for use as a medicament.